메뉴 건너뛰기




Volumn 280, Issue 5371, 1998, Pages 1940-1943

Gating of CaMKII by cAMP-regulated protein phosphatase activity during LTP

Author keywords

[No Author keywords available]

Indexed keywords

CYCLIC AMP; PHOSPHATASE;

EID: 0032546994     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.280.5371.1940     Document Type: Article
Times cited : (365)

References (35)
  • 1
    • 0027476024 scopus 로고
    • T. V. Bliss and G. L. Collingridge, Nature 361, 31 (1993); R. C. Malenka and R. A. Nicoll, Trends Neurosci. 16, 521 (1993).
    • (1993) Nature , vol.361 , pp. 31
    • Bliss, T.V.1    Collingridge, G.L.2
  • 3
    • 0028579743 scopus 로고
    • D. L. Pettit, S. Perlman, R. Malinow, Science 266, 1881 (1994); P. M. Lledo et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11175 (1995).
    • (1994) Science , vol.266 , pp. 1881
    • Pettit, D.L.1    Perlman, S.2    Malinow, R.3
  • 4
    • 0028880861 scopus 로고
    • D. L. Pettit, S. Perlman, R. Malinow, Science 266, 1881 (1994); P. M. Lledo et al., Proc. Natl. Acad. Sci. U.S.A. 92, 11175 (1995).
    • (1995) Proc. Natl. Acad. Sci. U.S.A. , vol.92 , pp. 11175
    • Lledo, P.M.1
  • 6
    • 0030952362 scopus 로고    scopus 로고
    • T. Abel et al., Cell 88, 615 (1997).
    • (1997) Cell , vol.88 , pp. 615
    • Abel, T.1
  • 8
  • 9
    • 2642606724 scopus 로고    scopus 로고
    • note
    • 35-thiophosphorylated I-1, T35A nonphosphorylatable I-1, autocamtide-3 and a related inactive control peptide (KKALHRQEAVDAL and KKALHAQERVDAL, respectively; gifts of A. P. Braun and H. Schulman) (28), and Rp-cAMPS were applied in the intracellular electrode and allowed to diffuse into the cell. In these experiments, HFS was delivered 40 to 60 min after impalement. For experiments in which Rp-cAMPS was applied in the superfusate, slices were exposed to 100 μM Rp-cAMPS in a maintenance chamber for 2 to 6 hours before recording. Synaptic stimulation consisted of monophasic, constant-current pulses of 100-μs duration delivered to the Schaffer collaterals (stratum radiatum in area CA3). A series of test pulses (four pulses at 0.2 Hz) was given every 5 or 10 min, and the excitatory postsynaptic potentials (EPSPs) within each series were averaged. EPSP amplitude and maximum initial slope (defined as the greatest slope within any 1-ms interval between the stimulus artifact and the EPSP peak) were measured offline for the averaged waveforms. LTP was induced by three trains of 100 pulses delivered at 100 Hz, separated by 10 min. The stimulus intensity used for HFS was adjusted to produce a 20-to 25-mV intracellular EPSP when measured from a membrane voltage of -80 mV; during HFS, the holding current was removed. Data were analyzed with either Student's t test or analyses of variance followed by Newman-Keuls post hoc comparisons. Summary data are presented as group means ± SEM.
  • 13
    • 0029870187 scopus 로고    scopus 로고
    • cDNAs encoding wild-type and nonphosphorylat-able (T35A) human I-1 were subcloned into the pT7-7 and pT7-5 vectors, respectively. The vectors were transformed into Escherichia coli BL21 (DE3). The recombinant proteins were expressed and purified by sequential trichloracetic acid (TCA) precipitation with I-1 purified from rabbit skeletal muscle as a marker. Homogeneously purified I-1 was obtained with preparative SDS-polyacrylamide gel electrophoresis (PAGE), and the purified proteins were phosphorylated with PKA and adenosine triphosphate (ATP)-γ-S [S. Endo, X. Zhou, J. Connor, B. Wang, S. Shenolikar, Biochemistry 35, 5220 (1996)].
    • (1996) Biochemistry , vol.35 , pp. 5220
    • Endo, S.1    Zhou, X.2    Connor, J.3    Wang, B.4    Shenolikar, S.5
  • 14
    • 2642678157 scopus 로고    scopus 로고
    • note
    • Field potentials were monitored and HFS delivered as described (7); 2 to 3 min after, the final train slices were removed from the recording chamber and placed on a cold plate. The CA1 region was rapidly dissected out and frozen at -70°C. Individual CA1 samples were lysed in 100 μl of lysis buffer [50 mM tris, 4 mM EGTA, 10 mM EDTA, 15 mM Na phosphate, 100 mM β-glycerophosphate, 10 mM NaF, 0.1 mM pepstatin, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 2 mM benzamidine (pH 7.5)] and ground three times for 30 s with a pellet pestle (Kontes glassware, Vineland, NJ). The resulting lysate was spun down at 15,000g at 4°C for 10 min, and the supernatant was then assayed for total protein concentration. Twenty micrograms of protein from each sample was run on a 15% SDS-PAGE gel. The samples were transferred onto a polyvinylidene difluoride membrane overnight (25-mA constant current) and then blotted with an antibody to either phosphorylated DARPP-32/I-1 or recombinant human I-1. The monoclonal antibody (mAb) to phosphorylated DARPP-32/I-1 (provided by G. L. Snyder and P. Greengard) (29) was blotted at 1/1000 dilution into tris-buffered saline (TBS) + 0.3% Tween 20. It was probed with a horseradish peroxidase (HRP)-conjugated antibody to mouse, diluted 1/2500 in TBS + 0.3% Tween. The polyclonal antibody to human I-1 was blotted at 1/2500 dilution in TBS and probed with 1/2500 dilution of antibody to rabbit. Both blots were developed with enhanced chemiluminescence (ECL), revealing bands at 28 kD, which correspond to native I-1 in these gels.
  • 15
    • 2642679793 scopus 로고    scopus 로고
    • note
    • Hippocampal slices were treated and dissected as described above (7, 12). Individual CA1 regions were homogenized in 50 mM tris (pH 7.5), 0.2 mM EDTA, 0.2 mM EGTA, leupeptin (2 μg/ml), aprotinin (2 μg/ml), and 10 nM okadaic acid. Phosphatase activity of 20 ng of protein of CA1 homogenate was measured with the Protein Phosphatase Assay System (GIBCO BRL) in a final concentration of 3.3 nM okadaic acid. With this method, inclusion of 100 nM thiophosphorylated I-1 inhibited >90% of phosphatase activity, confirming the selectivity of the system for PP1.
  • 18
    • 0028917370 scopus 로고
    • K. Fukunaga, D. Muller, E. Miyamoto, J. Biol. Chem. 270, 6119 (1995); A. P. Braun and H. Schulman, Annu. Rev. Physiol. 57, 417 (1995).
    • (1995) Annu. Rev. Physiol. , vol.57 , pp. 417
    • Braun, A.P.1    Schulman, H.2
  • 23
    • 0027395059 scopus 로고
    • 286-phosphorylated CaMKII α subunit (Affinity Bioreagents, Golden, CO) [B. L. Patton, S. S. Molloy, M. B. Kennedy, Mol. Biol. Cell 4, 159 (1993)] or a polyclonal antibody to CaMKII (Upstate Biotechnology, Lake Placid, NY). The mAb to autophosphorylated CaMKII was blotted at 1/2000 dilution into 5% nonfat dried milk, 0.1% Tween, and 1 μM microcystin-LR in phosphate-buffered saline (PBS). It was probed with an HRP-conjugated antibody to mouse at 1/3000 dilution into 5% nonfat dried milk and 0.1% Tween in PBS. The polyclonal antibody to CaMKII was blotted at 1 μg/ml in 3% nonfat dried milk in PBS and probed with a 1/3000 dilution of antibody to rabbit. Both blots were developed with ECL, revealing a band at ∼50 kD (corresponding to the α subunit of CaMKII).
    • (1993) Mol. Biol. Cell , vol.4 , pp. 159
    • Patton, B.L.1    Molloy, S.S.2    Kennedy, M.B.3
  • 26
    • 0015542144 scopus 로고
    • G. W. Sharp, Annu. Rev. Med. 24, 19 (1973); S. Traverse, N. Gomez, H. Paterson, C. Marshall, P. Cohen, Biochem. J. 288, 351 (1992).
    • (1973) Annu. Rev. Med. , vol.24 , pp. 19
    • Sharp, G.W.1
  • 33
    • 2642701446 scopus 로고    scopus 로고
    • note
    • Single-letter abbreviations for the amino acid residues are as follows: A, Ala; D, Asp; E, Glu; H, His; K, Lys; L, Leu; Q, Gln; R, Arg; and V, Val.
  • 35
    • 2642645130 scopus 로고    scopus 로고
    • note
    • Funded by grants from NIH (NS33646 to E.M.L., GM54508 to R.I., and DK52054 to S.S.) and the Veterans Administration (to E.M.L.). G.P.B. is supported by an individual postdoctoral National Research Service Award (GM18887). We thank H. Schulman and A. P. Braun for initial samples of autocamtide-3 and control peptide and for valuable discussions regarding phosphorylated-CaMKII antibodies.


* 이 정보는 Elsevier사의 SCOPUS DB에서 KISTI가 분석하여 추출한 것입니다.